Difference between revisions of "Part:BBa K3523005"

Revision as of 16:40, 26 October 2020

T7 pro-His-SOD-His-T7 ter

BBa_K3523005 contains BBa_K3523000, encoding the superoxide dismutases (SOD). SOD is a group of enzymes that catalyze the dismutation of superoxide radicals (O2−) to molecular oxygen (O2) and hydrogen peroxide (H2O2), providing cellular defense against reactive oxygen species.

Contribution and Biology

Our goal of this project is to construct an engineered bacteria which will scavenge superoxide compounds (ROS) in gut quickly and efficiently. To achieve it, we selected a classical enzymes -- superoxide dismutase (SOD), which are capable of effectively degrading ROS, for overexpression and purification in Escherichia coli BL21 (DE3). By monitoring ROS consumption, the ability of the engineered strain to degrade ROS was verified.

Figure 1

We use T7 promoter to start SOD protein transcription, and T7 terminator to end transcription. At the same time, insert a His protein tag into SOD protein for purification of SOD protein on the nickel column. This part can be used for topics related to the degradation of ROS in the future.

Engineering Success

Characterization of the biochemical characteristics of SOD:

SOD was expressed in Escherichia coli, bacterial cells were collected and broken, and SOD solution was obtained through isolation and purification, and further confirmed by the SDS-Page method, protein bands of the corresponding size were found (Fig.2).

Figure 2 SDS-Page assay the expression of SOD protein M: Protein Ladder; FT: Flow-through sample; W: Washing sample; 50: Elution sample with 50mM imidazole; 100: Elution sample with 100mM imidazole; 250: Elution sample with 250mM imidazole; 500: Elution sample with 500mM imidazole.

We used the classic nitroblue tetrazolium (NBT) color development method. Superoxide anion (O2-.) was produced by Xanthine and Xanthine Oxidase (XO) reaction system to reduce NBT to blue formazan, which had strong absorption at 560nm. While SOD can remove superoxide anions, so dirty formation is inhibited. The bluer the reaction solution is, the lower the activity of superoxide dismutase is, and vice versa. The activity level of superoxide dismutase can be calculated by colorimetric analysis. The detection principle is shown in Fig.3, and the detected absorbance is shown in Table.1.

Figure 2

Figure 3

The data is substituted into the formula for calculation:

Inhibition percentage=[(Ablank1-Ablank2) - (Asample-Ablank3)]/(Ablank1-Ablank2) * 100%=69.543%

Enzyme activity of sample=inhibition percentage / (1-inhibition percentage) (units)=2.283 U

Specific activity of SOD= enzyme activity of sample / amount of protein (units/mg)=1936.12 U/mg.

The results showed that SOD protein became dissolved in this E. coli expressing a condition, and the target protein is very pure. And SOD had excellent catalytic properties, which could successfully degrade ROS into H2O2

@@ Line 12: / Line 12: @@
 Characterization of the biochemical characteristics of SOD:
-SOD was expressed in Escherichia coli, bacterial cells were collected and broken, and SOD solution was obtained through isolation and purification, and further confirmed by the SDS-Page method, protein bands of corresponding size were found (Fig.2).
+SOD was expressed in Escherichia coli, bacterial cells were collected and broken, and SOD solution was obtained through isolation and purification, and further confirmed by the SDS-Page method, protein bands of the corresponding size were found (Fig.2).
 [[File:T--Xiamen_city-BBa_K3523006_figure 1.jpg|500px|thumb|center|Figure 2 SDS-Page assay the expression of SOD protein M: Protein Ladder; FT: Flow-through sample; W: Washing sample; 50: Elution sample with 50mM imidazole; 100: Elution sample with 100mM imidazole; 250: Elution sample with 250mM imidazole; 500: Elution sample with 500mM imidazole.]]
-We used the classic nitroblue tetrazolium (NBT) color development method. Superoxide anion (O2-.) was produced by Xanthine and Xanthine Oxidase (XO) reaction system to reduce NBT to blue formazan, which had strong absorption at 560nm.While SOD can remove superoxide anions, so dirty formation is inhibited. The bluer the reaction solution is, the lower the activity of superoxide dismutase is, and vice versa. The activity level of superoxide dismutase can be calculated by colorimetric analysis. The detection principle is shown in Fig.3, and the detected absorbance is shown in Table.1.
+We used the classic nitroblue tetrazolium (NBT) color development method. Superoxide anion (O2-.) was produced by Xanthine and Xanthine Oxidase (XO) reaction system to reduce NBT to blue formazan, which had strong absorption at 560nm. While SOD can remove superoxide anions, so dirty formation is inhibited. The bluer the reaction solution is, the lower the activity of superoxide dismutase is, and vice versa. The activity level of superoxide dismutase can be calculated by colorimetric analysis. The detection principle is shown in Fig.3, and the detected absorbance is shown in Table.1.
 [[File:T--Xiamen_city-BBa_K3523005_figure 2.jpg|500px|thumb|center|Figure 2]]
 [[File:T--Xiamen_city-BBa_K3523005_figure 3.jpg|500px|thumb|center|Figure 3]]
 The data is substituted into the formula for calculation:
 Inhibition percentage=[(Ablank1-Ablank2) - (Asample-Ablank3)]/(Ablank1-Ablank2) * 100%=69.543%
 Enzyme activity of sample=inhibition percentage / (1-inhibition percentage) (units)=2.283 U
 Specific activity of SOD= enzyme activity of sample / amount of protein (units/mg)=1936.12 U/mg.
-The results showed that SOD protein became dissolved in this E. coli expressing condition, and the target protein is very pure. And SOD had excellent catalytic properties, which could successfully degrade ROS into H2O2
+The results showed that SOD protein became dissolved in this E. coli expressing a condition, and the target protein is very pure. And SOD had excellent catalytic properties, which could successfully degrade ROS into H2O2